1. Field of the Invention
The present invention concerns: (a) methods for the rapid screening of drugs targeted to the V3 hypervariable loop of the human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2) envelope glycoproteins and (b) the use of these drugs for chemotherapy and prophylaxis of HIV-1 (or HIV-2) infections and the acquired immunodeficiency syndrome (AIDS).
2. Background Information
Antiviral drugs considered for therapy of Lentivirus infections can be classified according to: (1) the virus or cellular components they are targeted to (envelope glycoproteins and other structural components of the virion, virally-encoded enzymes, viral genes or their transcripts or specific cellular factors essential for virus replication) or (2) temporal stages in the virus replicative cycle, starting with attachment of the virus to cells, fusion with target cell membranes and virus uncoating (Mitsuya, H., Yarchoan, R., and Broder, S. (1990), "Molecular Targets for AIDS Therapy", Science, 249, 1533-1544; De Clercq, E. (1991), "Basic Approaches to Anti-retroviral Treatment", J. AIDS, 4, 207-218).
Drugs targeted to early steps in virus replication may have potential not only for immunotherapy of ongoing infections but also for prophylaxis, and thus may complement efforts to develop antiviral vaccines. Aurintricarboxylic acid (ATA), known as a potent inhibitor of protein-nucleic acid interactions (Gonzalez, R. G., Blackburn, B. J., Schleich, T. (1979), "Fractionation and Structural Elucidation of the Active Components of Aurintricarboxylic Acid, a Potent Inhibitor of Protein Nucleic Acid Interactions", Biochem. Biophys. Acta, 562, 534-545), was shown to inhibit the cytopathogenicity and replication of the human immunodeficiency virus type 1 (HIV-1) as measured by the production of the nucleocapsid protein P24 (Balzarini, J., Mitsuya, H., De Clercq E., Broder, S. (1986), "Aurintricarboxylic Acid and Evans Blue Represent Two Different Classes of Anionic Compounds Which Selectively Inhibit the Cytopathogenicity of Human T-cell Lymphotropic Virus Type III/Lymphadenopathy-associated Virus", Biochem. Biophys. Res. Comm., 136, 64-71).
Originally thought to inhibit HIV-1 replication by acting on the virus reverse transcriptase, it was concluded from subsequent studies that ATA also exerts its antiviral effects by attaching to the HIV-1 envelope glycoprotein gp120, thus inhibiting the interaction between HIV-1 virions and cellular CD4 receptors (Schols, D., Baba M., Pauwels, R., Desmyter, J., and De Clercq, E. (1989), "Specific Interaction of Aurintricarboxylic Acid With the Human Immunodeficiency Virus/CD4 Cell Receptor", Proc. Natl. Acad. Sci. USA. 86, 3322-3326; Cushman, M., Wang P., Chang, S. H., Wild, C., De Clercq, E., Schols, D., Goldman, M. E., and Bowen, J. A. (1991), "Preparation and anti-HIV Activities of Aurintricarboxylic Acid Fractions and Analogues: Direct Correlation of Antiviral Potency with Molecular Weight", J. Med. Chem., 34, 329-337).
It was reported several years ago that ATA inhibited the replication of HIV-1 in vitro (Balzarini et al., (1986), Biochem. Biohys. Res. Comm., 136, 64-71). Similar effects were observed with other polyanionic compounds having several benzene rings in their structure (Evans blue, suramine and fuchsin; Balzarini et al., (1986) supra; Baba, M., Schols, D., Pauwels, R., Balzarini, J., De Clercq, E., (1988), "Fuchsin Acid Selectively Inhibits Human Immunodeficiency Virus (HIV) Replication in vitro", Biochem. Biophys. Res. Commun., 155, 1404-1411). These compounds inhibited the reverse transcriptase of HIV-1, a possible mechanism for explaining their antiviral effect. This conclusion appears to be in agreement with the well established inhibitory effect of ATA on interactions between proteins and nucleic acids (Gonzalez et al, (1979), Biochem. Biophys. Acta, 562, 534-545). Commercially available ATA is prepared by treatment of salicylic acid with formaldehyde in the presence of sulfuric acid and sodium nitrite. The resulting product is a heterogeneous mixture containing monomeric ATA and a series of ATA polymers (Gonzalez et al., (1979), supra., Cushman et al., (1979), J. Med. Chem., 34, 329-337).
A direct correlation between the molecular mass of ATA polymers and their inhibitory effect on HIV-1 reverse transcriptase and on antiviral activity was established (Cushman et al., (1991); J. Med. Chem., 34, 329-337). The inhibitory activity of ATA on HIV-1 reverse transcriptase could be explained by binding of ATA (ATA polymers) to the enzyme protein.
Results of additional studies demonstrated that reverse transcriptase is not the only target for ATA. It was shown that ATA as well as Evans blue inhibited the interaction of the CD4 receptor for HIV-1, expressed on the surface of cells, with gp120 or with anti-CD4 monoclonal antibodies (Schols et al., (1989), Proc. Natl. Acad. Sci. USA, 86, 3322-3326; Schols et al, (1990), Virology, 175, 556-561; Weaver, J. L., Gergely, P., Pine, P. S., Patzer, E., Aszalos, A. (1990), "Polyionic Compounds Selectively Alter Availability of CD4 Receptors for HIV Coat Protein rgp120, AIDS Res. & Hum. Retro., 6, 1125-1130).
However, ATA is an inefficient inhibitor of the reaction between recombinant gp120 and recombinant CD4, and complete inhibition of this reaction can be observed only at high concentrations of ATA (.apprxeq.1 mg/ml).
Several polyanionic substances (dextran sulfate, pentosan polysulfate, heparin, Evans blue, suramin and ATA) were also reported to inhibit the reaction of gp120 expressed on the surface of HIV-1-infected cells with monoclonal anti-gp120 antibodies (Schols et al., (1990), Virology, 175, 556-561; De Clercq, (1991), J. AIDS. 4, 207-218). These results suggest the occurrence of multiple target sites for ATA and for other polyanionic compounds on HIV-1 proteins. The availability of a multitude of antibodies with predetermined specificity, generated by immunization with synthetic peptides derived from the entire sequence of HIV-1 gp120/gp41, as reported in (Neurath et al., (1990), Molec. Immunol., 27, 539-549; Neurath and Strick, (1990), J. Gen. Virol., 71, 85-95) offers the possibility to delineate precisely the target site(s) for ATA on HIV-1 gp120.
The gp120 V3 hypervariable loop is discussed in the following publications: Goudsmit, J. (1988), "Immunodominant B-cell Epitopes of the HIV-1 Envelope Recognized by Infected and Immunized Hosts," AIDS, 2, S41-S45; Goudsmit, J., Debouck, C., Meloen, R. H., Smit, L., Bakker, M., Asher, D. M., Wolff, A. V., Gibbs Jr., C. J., and Gajdusek, D. C. (1988), "Human Immunodeficiency Type 1 Neutralization Epitope with Conserved Architecture Elicits Early Type-specific Antibodies in Experimentally Infected Chimpanzees", Proc. Natl. Acad. Sci. USA, 85, 4478-4482; Goudsmit, J., Kuiken, C. L., and Nara, P. L. (1989), "Linear Versus Conformational Variation of V3 Neutralization Domains of HIV-1 During Experimental and Natural Infection", AIDS, 3, S119-S123; Linsley, P. S., Ledbetter, J. A. , Kinney-Thomas, E., and Hu, S. L. (1988), "Effects of Anti-gp120 Monoclonal Antibodies on CD4 Receptor Binding by the env Protein of Human Immunodeficiency Virus Type 1", J. Virol., 62, 3695-3702; Matsushita, S., Robert-Guroff, M., Rusche, J., Koito A., Hattori, T., Hoshino, H., Javaherian, K., Takatsuki, K., and Putney, S. D. (1988), "Characterization of a Human Immunodeficiency Virus Neutralizing Monoclonal Antibody and Mapping of the Neutralizing Epitope", J. Virol., 62, 2107-2114; Palker, T. J., Clark, M. E., Langlois, A. J., Matthews, T. J., Weinhold, K. J., Randall, R. R., Bolognesi, D. P. and Haynes, B. F. (1988), "Type-Specific Neutralization of the Human Immunodeficiency Virus with Antibodies to env-encoded Synthetic Peptides", Proc. Natl. Acad. Sci. USA, 85, 1932-1936; Rusche, J. R., Javaherian, K., McDanal, C., Petro, J., Lynn, D. L., Grimaila, R., Langlois, A., Galo, R. C., Arthur, L. O., Fischinger, P. J., Bolognesi, D. P., Putney, S. D. and Matthews, T. J. (1988), "Antibodies that Inhibit Fusion of Human Immunodeficiency Virus-infected Cells Bind a 24-amino Acid Sequence of the Viral Envelope, gp120", Proc. Natl. Acad. Sci. USA, 85, 3198-3202; Skinner, M. A., Langlois, A. J., McDanal, C. B., McDougal, J. S., Bolognesi, D. P., and Matthews, T. J. (1988), "Neutralizing Antibodies to an Immunodominant Envelope Sequence do not Prevent gp120 Binding to CD4", J. Virol., 62, 4195-4200; Kenealy, W. R., Matthews, T. J., Ganfield, M. C., Langlois, A. J., Waselefsky, D. M., and Petteway, Jr. S. R. (1989), "Antibodies from Human Immunodeficiency Virus-infected Individuals Bind to a Short Amino Acid Sequence that Elicits Neutralizing Antibodies in Animals," AIDS Res. Human Retroviruses, 5, 173-182; Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Putney, S. D., and Matthews, T. J. (1989), "Principal Neutralizing Domain of the Human Immunodeficiency Virus type 1 Envelope Protein, Proc. Natl. Acad. Sci. USA, 86, 6768-6772; Javaherian, K., Langlois, A. J., LaRosa, G. J., Profy, A. T., Bolognesi, D. P., Herlihy, W. C., Putney, S. C., Matthews, T. J. (1990), "Broadly Neutralizing Antibodies Elicited by the Hypervariable Neutralizing Determinant of HIV-1", Science, 250, 1590-1593; Fung, M. S. C., Sun, C. R. Y., Liou, R. S., Gordon, W.; Chang, N. T., Chang, T. W., and Sun, N. C. (1990), "Monoclonal Anti-idiotypic Antibody Mimicking the Principal Neutralization Site in HIV-1 gp120 Induces HIV-1 Neutralizing Antibodies in Rabbits" J. Immunol., 145, 2199-2206; Hart, M. K., Palker, T. J. Matthews, T. J., Langlois, A. J., Lerche N. W., Martin, M. E. Scearce, R. M., McDanal, C., Bolognesi, D. P., and Haynes, B. F. (1990), "Synthetic Peptides Containing T and B Cell Epitopes From Human Immunodeficiency Virus Envelope gp120 Induce anti-HIV Proliferative Responses and High Titers of Neutralizing Antibodies in Rhesus Monkeys., J. Immunol., 145, 2677-2685; Neurath et al, (1990), J. Gen. Virol., 71, 85-95; Profy, A. T., Salinas, P. A., Eckler, L. I., Dunlop, N. M., Nara, P. L., and Putney, S. C., (1990), "Epitopes Recognized by the Neutralizing Antibodies of an HIV-1-infected Individual", J. Immunol., 144, 4641-4647 and Scott, Jr., C. F., Silver, S., Profy, A. T., Putney, S. D., Langlois A., Weinhold, K., and Robinson, J. E. (1990), "Human Monoclonal Antibody that Recognizes the V3 Region of Human Immunodeficiency Virus gp120 and Neutralizes the Human T-Lymphotropic Virus type III.sub.MN Strain," Proc. Natl. Acad. Sci. USA. 87, 8597-8601.
PCT WO 89/11277 discusses compositions for the inhibition of replication of human immunodeficiency virus, containing one or more porphyrins possessing antiviral activity.